Supporting Information for:

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1 Supporting Information for: Indocyanine-Based Activatable Fluorescence Turn-On Probe for -Glutamyltranspeptidase and Its Application to the Mouse Model of Colon Cancer Seokan Park, a Soo-Yeon Lim, a Sang Mun Bae, b,c Sang-Yeob Kim, b,c Seung-Jae Myung, *b,d Hae-Jo Kim *a a Department of Chemistry, Hankuk University of Foreign Studies, Yongin , Republic of Korea. Tel.: ; Fax: ; haejkim@hufs.ac.kr (H.-J. Kim) b Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul , Republic of Korea c Department of Medicine, University of Ulsan College of Medicine, Seoul , Republic of Korea d Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul , Republic of Korea. Tel.: ; Fax: ; sjmyung@amc.seoul.kr (S.-J. Myung) Contents 1. NMR spectra S2 2. Mass spectra S5 3. LOD and Titration S8 4. ph profile S9 5. Inhibitor assay S1 6. HPLC profile S11 7. Cell viability assay S12 S1

2 5 1. NMR spectra ppm (t1) Fig. S1. ppm 4 (t1) Hz and 1 Hz spectra of 1 in CD 3 OD ppm (f1) Fig. S2. ppm 4 (t1) Hz and 1 Hz spectra of 2 in CD 3 OD. 1 5 S2

3 ppm (f1) Fig. S3. ppm 4 (t1) Hz and 1 Hz spectra of 3 in CD 3 OD ppm (f1) Fig. ppm (t1) S4. 4 Hz and 1 Hz spectra of AP-Glu in CD 3 OD. 1 5 S3

4 Synthesis of AP To a solution of 1. mmol of 4-hydroxybenzaldehyde, 1. mmol of 1-(5-carboxypentyl)-2,3,3-trimethyl- 3H-indolium bromide in 2 ml of EtOH was stirred at 8 o C overnight. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by chromatography on silica gel (DCM-MeOH as eluent, 1:1 v/v) to afford 238 mg of AP in 52 % yield as a red solid. 1 H NMR (4 MHz, CD 3 OD): 8.4 (d, J = 16., 1H), 8.9 (d, J = 8.8, 2H), (m, 2H), (m, 2H), 7.39 (d, J = 16., 1H), 6.95 (d, J = 8.8, 2H), 4.59 (t, J = 7.2), 2.27 (t, J = 7.2, 2H), (m, 2H), 1.85 (s, 6H), (m, 2H), (m, 2H). 13 C NMR (1 MHz, CD 3 OD): , 18.5, , , , , , 13.45, 13.12, 127.6, 124.2, , 115.5, 18.78, 53.31, 47.43, 36.69, 29.3, 27.37, 27.16, ppm (t1) Fig. S5. ppm 4 (f1) Hz and 1 Hz spectra of AP in CD 3 OD. 1 5 S4

5 2. Mass spectra Fig. S6. Low resolution of 1 (MALDI +, DHB): m/z found calcd for C 21 H 32 N 2 O 6 Na ([M+Na] + ) Fig. S7. MALDI-TOF mass spectra of 2 (MALDI +, DHB): m/z found calcd for C 28 H 36 N 2 O 7 Na ([M+Na] + ). S5

6 Fig. S8. Low (MALDI +, DHB) and high resolution mass spectra of 3 (FAB +, m-nba): m/z found , calcd for C 45 H 59 N 3 O 8 ([M+H] + ). S6

7 Fig. S9. Low and high resolution mass spectra of AP-Glu (FAB +, m-nba): m/z found , calcd for C 36 H 42 N 3 O 6 ([M] + ). S7

8 3. Limit of detection (LOD) and Titration F 557 nm 2 1 y =.2416x R 2 = U/L Fig. S1. Fluorescence intensity of GGT was measured between and 5 U/L of GGT. [AP-Glu] = 2 M in PBS buffer (1 X, ph 7.4). Excitation wavelength: 461 nm. Determination of the detection limit: First the calibration curve was obtained from the plot of fluorescence intensity as a function of the analyte concentration (GGT). The regression curve equation was then obtained for the lower concentration part. The detection limit = 3 S.D. / m where m is the slope of the linear equation, and S.D. represents the standard deviation for the fluorescence intensity ratio of the assay system in the absence of GGT. 557nm = [GGT] (R =.995) LOD = 3.12 /.3979 =.15 U/L 4 3 F 557 nm U/L Fig. S11. Titration of GGT was measured between and 5 U/L of GGT. [AP-Glu] = 2 M in PBS buffer (1 X, ph 7.4). Excitation wavelength: 461 nm. S8

9 Flu at 557nm 4. ph profile AP-Glu AP ph Fig. S12. ph profile of AP-Glu and AP (2 M). Excitation wavelength: 461 nm S9

10 5. Inhibitor assay A 525 /A AP-Glu AP-Glu+gGT AP-Glu+gGT+GGsTop time/h Fig. S13. Inhibitor assay of AP Glu (2 M in PBS, ph 7.4) in presence of GT (5 unit/l) and an enzyme inhibitor (GGsTop TM, 2 mm). S1

11 6. HPLC profile D C 5 A424nm B 1 Time / min T R Fig. S14. HPLC profile (OPTIMAPAK, MeOH:H 2 O = 43:57, 2 ml/min) of AP-Glu (1 mm, t R = 15 min) in presence of 5 U/mL of GT in PBS buffer (1X, ph 7.4). (A) AP-Glu + GT after min, (B) AP-Glu + GT after 1 min, (C) AP-Glu+ GT after 5 min, (D) AP (1 mm, t R = 9 min). A S11

12 Cell viability (%) 7. Cell viability assay Cell viability assay. HCT116 cells were plated in 1 l of media at 6, cells per well onto white clear-bottom 96-well plates (Corning Costar, NY, USA). Cells were incubated for 24h and 48h with or without compound before carrying out the viability assay. Using the Cell Titer-Glo Luminescent Cell Viability assay kit (Promega, WI, USA) and instructions, luminescent measurements were taken on an EnSpire 23 multilabel reader (Perkin Elmer, MA, USA). The graphically represented values are means s.d. for three independent samples h treatment 48h treatment DMSO Fig. S15. Viability of HCT116 cells treated with AP-Glu, where HCT116 cells were treated with AP-Glu or control (DMSO), and cell viability was determined by Cell Titer-Glo assay after incubation for 24h or 48 h. S12